Hi,
I am doing a western blot of SIRT1 but don't have much experience in SIRT1 western blotting.
I am working with a rabbit polyclonal anti-SIRT1 antibody (abcam, ab7343) at 1 µg/mL on mouse kidney extracts. The predicted size is 81 kDa and the observed size is 120 kDa, according to the manufacturer.
I have used 7% tris-acetate gels and PVDF membranes to make this blot (attached document).
As you can see I get multiple bands and am wondering which one I should consider for SIRT1 semi-quantification. The strong band on the bottom of the picture (slightly < 80kDa) should be an "inactive fragment", then there is a smear (degradation of SIRT1?). Finally, I get two distinct bands, between 110 and 160 kDa, so they probably are what I am looking for, but I don't know which one is the most relevant. Is the upper one an "activated" form of SIRT1 (through phosphorylation, acetylation, sumoylation, methylation etc.) or is it something else?
Thank you very much,
Thibault Teissier
As the abcam website, specifically for your antibody, shows that SIRT1 band is above 100KDa, technically, the brightest band should be the above 100KDa. In your case, I assume the 80KDa band is either degraded SIRT1 or non-specific band. I suggest to run another WB with careful protein extraction (add protease and phosphatase inhibitors) and use a little harsh washing process like TSBT with at least 4x (5min each). Hope you will get the right band. Good luck
Hi,
Thank you for you answers.
I actually already extract my proteins with protease and phosphatase inhibitors. Maybe I should order new ones in case they are not active anymore?
I usually wash my membranes with TBST (3x10 minutes) and block the membranes 1h-1h30 with 5% milk.
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